Retroviruses are classified in several ways. They are divided into various groups on the basis of their morphology. These groups are A,B,C and D type viruses. They are also classified as belonging to one of three subfamilies, namely oncoviruses, spumaviruses and lentiviruses.
Mason-Pfizer Monkey Virus (MPMV) is a D-type retrovirus first discovered in breast carcinoma tissue from a rhesus monkey. Despite this, and its classification into the oncovirus subfamily, it does not contain an identified oncogene and there is no evidence that it has oncogenic potential.
D-type viruses are distinguished from other retrovirus families such as the C-type viruses. The latter are characterised by capsid assembly at the cell membrane, and include viruses of the lentivirus group, e.g. Human Immunodeficiency Virus (HIV). Morphologically, in their core structure, D-type viruses also differ from B-type viruses such as Mouse Mammary Tumor Virus (MMTV). Thus the D-type viruses are completely distinct from these other types and as an example of this distinction they have specific signals in the form of amino-acid signal sequences in their assembled ICAP which facilitate transport of the ICAP to the cell membrane. For this reason and others they must be considered as a separate group with unique cis and trans-acting regulatory signals. MPMV is also distinguished from C-type viruses by the ability of the virus envelope to activate the human complement system. This leads to lysis of the virus.
Retroviruses are RNA viruses which replicate through a DNA proviral intermediate which is integrated in the genome of the infected host cell. The virion particle contains a dimer of positive-strand genomic RNA molecules. This genomic RNA is the full-length species transcribed from the proviral DNA by the host RNA polymerase II. A proportion of these full length RNAs which encode the gag and pol genes of the virus are translated by the host cell ribosomes, to produce the structural and enzymic proteins required for production of virion particles. The provirus also gives rise to a variety of smaller singly and multiply-spliced mRNAs coding for the envelope proteins and, in the case of more complex retroviruses, a group of regulatory proteins. The genomic (and subgenomic) RNA molecules are structurally similar to cellular mRNAs in having a 5' m.sup.7 G cap and a polyadenylated 3' tail.
A series of problems must be addressed for successful packaging of genomic RNA:
The full-length RNA must be packaged preferentially over the spliced viral messages as it is the only one carrying the full complement of genetic information for the next generation of virions. The virus must also specifically select the genomic RNA against the enormous quantity and variety of physically similar host cell mRNAs as, unlike many other viruses, retroviruses do not generally arrest host RNA synthesis. There must be a mechanism whereby genomic RNA to be packaged is recognised such that a proportion is either protected from being translated and transported to an assembly site or is associated with the gag precursor polyprotein for which it has just coded immediately after translation. Lastly, there is the stoichiometric problem of having to package the correct number of genomes in association with 3-4000 gag precursor proteins, adequate numbers of reverse transcriptase molecules, a protease, tRNA primers and, in some cases, multiple copies of regulatory proteins.
Packaging the genome thus entails problems of specificity of selection of RNA and also considerations of RNA compartmentalisation.
The virus overcomes these problems by the presence of cis-acting elements, i.e. "packaging signals", in the viral genomic MRNA. Studies on spontaneously arising and laboratory constructed viral mutants have confirmed that specific sequences are critical for RNA recognition and encapsidation. Linial et al, Cell 15:1371-1381 (1978); Mann et al, Cell 33:153-159 (1983); Watanabe et al, PNAS USA 79:5986-5990 (1979) and WO-A-9119798 disclose that deletions in the 5' untranslated leader sequence lead to defects in packaging in, respectively, Rous Sarcoma Virus (RSV), Moloney Murine Leukemia Virus (MoMLV), Spleen Necrosis Virus (SNV) and HIV.
Deletion mutants have defined sequences necessary for RNA packaging in several retroviruses. In some of these, the extent of the sequence sufficient for packaging has also been mapped. Implicit in the description of packaging signals and RNA secondary structure is the premise that, if this sequence is introduced into heterologous RNA then, theoretically, the heterologous RNA should be encapsidated by retroviral particles. Constraints on packaging include the theoretical one (for which Mann et al, J. Virol. 54:401-407 (1985) provide some circumstantial evidence) that sequences adjacent to the packaging signal (PSI) should not favour the formation of alternative secondary structures disrupting PSI. Additionally, the total length of RNA packaged is physically limited by the capacity of the virus to package RNA of a certain size. In HIV, proviral constructs incorporating heterologous genes have been shown by Terwilliger et al, PNAS USA 86:3857-3861 (1989) to lead to a replication defect when the total length of the viral RNA produced significantly exceeds that of the original virus. The replication defect is consistent with a declining efficiency of RNA packaging.
Nevertheless, there is significant variability between different viruses in the nature and site of their encapsidation sequences. The mechanism of RNA recognition is so poorly understood that theoretically it is not possible to make predictions of the exact site and nature of encapsidation sequences without experimental data. There is none for D-type retroviruses.
The development of retroviral vector systems has been a direct development of the work described above. In these systems, a packaging-defective "helper" virus is used to generate particles which encapsidate a highly modified RNA genome (the vector). Watanabe et al, Mol. Cell Biol. 3:2241-2249 (1983), and Eglitis et al, BioTechniques 6:608-614 (1988), report that vectors containing a minimum of the viral long terminal repeats, the packaging signal and a primer-binding site together with a heterologous marker gene have been encapsidated into virion particles and transferred to the cells for which the parent virus is tropic. By this means, it has been possible to define the minimal sequence required for encapsidation of RNA into a virus particle.
Adam et al, J. Virol. 62:3802-3806 (1988), disclose that, for MoMLV, the sequence sufficient for packaging encompasses the 5' leader region first defined by deletion as leading to a packaging defect. No additional sequences were essential although gag sequences enhanced packaging of the vector.
WO-A-9119798 discloses that an HIV-based vector containing essentially only the 5' leader sequence as a potential packaging signal was reported to be successfully encapsidated by an HIV-based packaging system. This work has not yet been confirmed. Indeed, there has been failure, confirmed herein, to encapsidate HIV RNA containing only the 5' leader sequence.